Identification of novel antimicrobial agents using membrane potential indicator dyes

ABSTRACT

Novel screening methods for identifying antimicrobial agents involving use of membrane potential indicator dyes are provided.

This application is a continuation of U.S. Ser. No. 09/404,926, filedSep. 24, 1999, which claims priority based on U.S. ProvisionalApplication Serial No. 60/143,485 filed Jul. 12, 1999, U.S. ProvisionalApplication Serial No. 60/109,905 filed Nov. 25, 1998, and U.S.Provisional Application Serial No. 60/101,778 filed Sep. 25, 1998.

FIELD OF THE INVENTION

The invention relates generally to screening methods involving use ofmembrane potential indicator dyes for identifying antimicrobial agents,including antifungal and antibacterial compounds.

BACKGROUND OF THE INVENTION

Fungi are not only important human and animal pathogens, but they arealso among the most common causes of plant disease. Fungal infections(mycoses) are becoming a major concern for a number of reasons,including the limited number of antifungal agents available, theincreasing incidence of species resistant to known antifungal agents,and the growing population of immunocompromised patients at risk foropportunistic fungal infections, such as organ transplant patients,cancer patients undergoing chemotherapy, burn patients, AIDS patients,or patients with diabetic ketoacidosis. The incidence of systemic fungalinfections increased 600% in teaching hospitals and 220% in non-teachinghospitals during the 1980's. The most common clinical isolate is Candidaalbicans (comprising about 19% of all isolates). In one study, nearly40%of all deaths from hospital-acquired infections were due to fungi.[Sternberg, Science, 266:1632-1634 (1994).]

Known antifungal agents include polyene derivatives, such asamphotericin B (including lipid or liposomal formulations thereof) andthe structurally related compounds nystatin and pimaricin; flucytosine(5-fluorocytosine); azole derivatives (including ketoconazole,clotrimazole, miconazole, econazole, butoconazole, oxiconazole,sulconazole, tioconazole, terconazole, fluconazole, itraconazole,voriconazole [Pfizer] and SCH56592 [Schering-Plough]);allylamines-thiocarbamates (including toinaftate, naftifine andterbinafine); griseofulvin; ciclopirox; haloprogin; echinocandins(including MK-0991 [Merck]); and nikkomycins. Recently discovered asantifungal agents are a class of products related tobactericidal/permeability-increasing protein (BPI), described in U.S.Pat. Nos. 5,627,153, 5,858,974, 5,652,332, 5,763,567 and 5,733,872, thedisclosures of all of which are incorporated herein by reference.

Resistance of bacteria and other pathogenic organisms to antimicrobialagents is an increasingly troublesome problem. The acceleratingdevelopment of antibiotic-resistant bacteria, intensified by thewidespread use of antibiotics in farm animals and overprescription ofantibiotics by physicians, has been accompanied by declining researchinto new antibiotics with different modes of action. [Science, 264:360-374 (1994).]

Gram-positive bacteria have a typical lipid bilayer cytoplasmic membranesurrounded by a rigid cell wall that gives the organisms theircharacteristic shape, differentiates them from eukaryotic cells, andallows them to survive in osmotically unfavorable environments. Thiscell wall is composed mainly of peptidoglycan, a polymer ofN-acetylglucosamine and N-acetylmuramic acid. In addition, the cellwalls of gram-positive bacteria contain teichoic acids which areanchored to the cytoplasmic membrane through lipid tails, giving rise tolipoteichoic acids. The various substituents on teichoic acids are oftenresponsible for the biologic and immunologic properties associated withdisease due to pathogenic gram-positive bacteria. Most pathogenicgram-positive bacteria have additional extracellular structures,including surface polysaccharides, capsular polysaccharides, surfaceproteins and polypeptide capsules.

Gram-negative bacteria also have a cytoplasmic membrane and apeptidoglycan layer similar to but reduced from that found ingram-positive organisms. However, gram-negative bacteria have anadditional outer membrane that is covalently linked to the tetrapeptidesof the peptidoglycan layer by a lipoprotein; this protein also containsa special lipid substituent on the terminal cysteine that embeds thelipoprotein in the outer membrane. The outer layer of the outer membranecontains the lipopolysaccharide (LPS) constituent.

Antibacterial agents are generally directed against targets not presentin mammalian cells. One major difference between bacterial and mammaliancells is the presence in bacteria of a rigid wall external to the cellmembrane. Thus, chemotherapeutic agents directed at any stage of thesynthesis, export, assembly, or cross-linking of peptidoglycan lead toinhibition of bacterial cell growth and, in most cases, to cell death.These agents include bacitricin, the glycopeptides (vancomycin andteichoplanin), β-lactam antibiotics (penicilins, cephalosporins,carbapenems, and monobactams). Virtually all the antibiotics thatinhibit bacterial cell wall synthesis are bactericidal. However, much ofthe loss of cell wall integrity following treatment with cellwall-active agents is due to the bacteria's own cell wall-remodelingenzymes (autolysins) that cleave peptidoglycan bonds in the normalcourse of cell growth. In the presence of antibacterial agents thatinhibit cell wall growth, autolysis proceeds without normal cell wallrepair; weakness and eventually cellular lysis occur. There are alsoantibacterial agents that do not affect cell wall synthesis but insteadare believed to alter cell membrane permeability, such as the polymyxins(polymyxin B and colistin, or polymyxin E) and gramicidin A.

Another group of antibacterial agents are those that inhibit proteinsynthesis; most of these interact with the bacterial ribosome. Thedifference between the composition of bacterial and mammalian ribosomesgives these compounds their selectivity. These agents include theaminoglycosides (e.g., gentamicin, kanamycin, tobramycin, streptomycin,netilmicin, neomycin, and amikacin), the macrolides (e.g., erythromycin,clarithromycin, and azithromycin), the lincosamides (e.g., clindamycinand lincomycin), chloramphenicol, the tetracyclines (e.g., tetracycline,doxycycline, and minocycline) and mupirocin (pseudomonic acid).

Another group of antibacterial agents are antimetabolites that interferewith bacterial synthesis of folic acid. Inhibition of folate synthesisleads to cessation of cell growth and, in some cases, to bacterial celldeath. The principal antibacterial antimetabolites are sulfonamides(e.g., sulfisoxazole, sulfadiazine, and sulfamethoxazole) andtrimethoprim.

Yet a further group of antibacterial compounds affects nucleic acidsynthesis or activity. These agents include the quinolones (e.g.,nalidixic acid and its fluorinated derivatives norfloxacin,ciprofloxicin, ofloxacin, and lomofloxacin), which inhibit the activityof the A subunit of DNA gyrase, rifampin, nitrofurantoin, andmetronidazole (which not only has activity against the electrontransport system but also is believed to cause DNA damage).

BPI protein products are also described to have antibacterial activitiesin U.S. Pat. Nos. 5,198,541 and 5,523,288 and International PublicationNo. WO 95/08344 (PCT/US94/11255), all of which are incorporated byreference herein, disclosing activity against gram-negative bacteria,and U.S. Pat. Nos. 5,578,572 and 5,783,561 and International PublicationNo. WO 95/19180 (PCT/US95/00656), all of which are incorporated byreference herein, disclosing activity against gram-positive bacteria andmycoplasma, and co-owned, co-pending U.S. application Ser. No.08/626,646, which is in turn a continuation of U.S. application Ser. No.08/285,803, which is in turn a continuation-in-part of U.S. applicationSer. No. 08/031,145 and corresponding International Publication No. WO94/20129 (PCT/US94/02463), all of which are incorporated by referenceherein, disclosing activity against mycobacteria.

BPI protein products have been shown to have additional antimicrobialactivities. For example, U.S. Pat. No. 5,646,114 and InternationalPublication No. WO 96/01647 (PCT/US95/08624), all of which areincorporated by reference herein, disclose activity of BPI proteinproducts against protozoa.

Bactericidal/permeability-increasing protein (BPI) is a protein isolatedfrom the granules of mammalian polymorphonuclear leukocytes (PMNs orneutrophils), which are blood cells essential in the defense againstinvading microorganisms. See Elsbach, 1979, J. Biol. Chem., 254: 11000;Weiss et al., 1987, Blood 69: 652; Gray et al., 1989, J. Biol. Chem.264: 9505. The amino acid sequence of the entire human BPI protein andthe nucleic acid sequence of DNA encoding the protein (SEQ ID NOS: 2 and3) have been reported in FIG. 1 of Gray et al., J. Biol. Chem., 264:9505(1989), incorporated herein by reference. Recombinant human BPIholoprotein has also been produced in which valine at position 151 isspecified by GTG rather than GTC, residue 185 is glutamic acid(specified by GAG) rather than lysine (specified by AAG) and residue 417is alanine (specified by GCT) rather than valine (specified by GTT). AnN-terminal fragment of human BPI possesses the anti-bacterial efficacyof the naturally-derived 55 kD human BPI holoprotein. (Ooi et al., 1987,J. Bio. Chem. 262: 14891-14894). In contrast to the N-terminal portion,the C-terminal region of the isolated human BPI protein displays onlyslightly detectable anti-bacterial activity against gram-negativeorganisms and some endotoxin neutralizing activity. (Ooi et al., 1991,J. Exp. Med. 174: 649). An N-terminal BPI fragment of approximately 23kD, referred to as rBPI₂₃, has been produced by recombinant means andalso retains anti-bacterial including anti-endotoxin activity againstgram-negative organisms (Gazzano-Santoro et al., 1992, Infect. Immun.60: 4754-4761). An N-terminal fragment analog designated rBPI₂₁ has beendescribed in co-owned, co-pending U.S. Pat. No. 5,420,019.

Three separate functional domains within the recombinant 23 kDN-terminal BPI sequence have been discovered (Little et al., 1994, J.Biol. Chem. 269: 1865). These functional domains of BPI designateregions of the amino acid sequence of BPI that contributes to the totalbiological activity of the protein and were essentially defined by theactivities of proteolytic cleavage fragments, overlapping 15-merpeptides and other synthetic peptides. Domain I is defined as the aminoacid sequence of BPI comprising from about amino acid 17 to about aminoacid 45. Initial peptides based on this domain were moderately active inboth the inhibition of LPS-induced LAL activity and in heparin bindingassays, and did not exhibit significant bactericidal activity. Domain IIis defined as the amino acid sequence of BPI comprising from about aminoacid 65 to about amino acid 99. Initial peptides based on this domainexhibited high LPS and heparin binding capacity and exhibitedsignificant antibacterial activity. Domain III is defined as the aminoacid sequence of BPI comprising from about amino acid 142 to about aminoacid 169. Initial peptides based on this domain exhibited high LPS andheparin binding activity and exhibited surprising antimicrobialactivity, including antifungal and antibacterial (including, e.g.,anti-gram-positive and anti-gram-negative) activity. The biologicalactivities of peptides derived from or based on these functional domains(i.e., functional domain peptides) may include LPS binding, LPSneutralization, heparin binding, heparin neutralization or antimicrobialactivity.

Of interest to the background of the present invention are dyeindicators of membrane potential which have been available for manyyears and have been employed to study cell physiology. Thesepotentiometric dyes are organic compounds whose spectral properties aresensitive to changes in membrane potential. They can be classifiedgenerally into “fast” dyes, which can follow changes in potential in themillisecond range, and “slow” dyes, which generally operate by apotential-dependent partitioning between the extracellular medium andeither the membrane or the cytoplasm. This partitioning of slow dyesoccurs by redistribution of the dye via interaction of the voltagepotential with ionic charge on the dye. Slow dyes include three generalchromophore types: cyanines [such as Di-O-C6(3) and Di-S-C2(5)], oxonols[such as oxonol-VI and DiS-BaC2(3)] and rhodamines [such asrhodamine-123 and TMRE JPW-179]. [See Loew, Chapter 8 in BiomembraneElectrochemistry, Blank and Vodyanoy, eds., American Chemical Society,Washington, D.C. (1994), pages 151-173.]

The cyanine class of dyes are symmetrical molecules with delocalizedpositive charges. Depending on the nature of the dye and itsconcentration, the potential-dependent uptake can produce either anincrease or a decrease in fluorescence intensity. In general,accumulation of the dye and membrane binding leads to enhancement offluorescence. At high lipid-dye ratios, however, many of the cyaninedyes tend to aggregate, resulting in fluorescence self-quenching. Mostcarbocyanine dyes with short (C1-C6) alkyl chains stain mitochondria oflive cells when used at low concentrations (˜0.5 μM or ˜0.1 μg/mL);those with pentyl or hexyl substituents also stain the endoplasmicreticulum when used at higher concentrations (˜5-50 μM or ˜1-10 μg/mL).The cyanine dye DiOC₆(3) (3,3′-dihexyloxacarbocyanine iodide) has lesstendency to aggregate and displays an increased fluorescence quantumyield as it binds to the subcellular membranes. DiOC₆(3) is lipophilicand is often used as a stain for mitochondria and endoplasmic reticulumin eukaryotic cells.

The green fluorescent cyanine dye JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyaninehalide, available as an iodide from Molecular Probes or as a chloridefrom Biotium, Inc.) exists as a monomer at low concentrations or at lowmembrane potential. However, at higher concentrations (aqueous solutionsabove 0.1 μM) or at higher potentials, JC-1 forms red fluorescent“J-aggregates ” that exhibit a broad excitation spectrum of 485 to 585nm and an emission maximum at ˜590 nm. Emission from this dye has beenused to investigate mitochondrial potentials in live cells byratiometric techniques. Various types of ratio measurements are possibleby combining signals from the monomer (absorption/emission maxima˜510/527 nm in water) and the J-aggregate. Optical filters designed forfluorescein and tetramethylrhodamine can be used to separately visualizethe monomer and J-aggregate forms, respectively, or both forms can beobserved simultaneously using a standard fluorescein longpass opticalfilter set.

The oxonols are anionic molecules that also show enhanced fluorescenceupon binding to membranes. However, because of their negative charge,binding of oxonols is promoted by depolarization of the membrane. Thenegative charge of oxonols also lessens intracellular uptake and reducestheir association with intracellular organelles.

Rhodamine-123 is a cell-permeant, cationic, fluorescent dye that isreadily sequestered by active mitochondria without inducing cytotoxiceffects. Uptake and equilibration of rhodamine-123 is rapid (a fewminutes) compared to dyes such as DASPMI, which may take 30 minutes orlonger, and this dye is especially suited for flow cytometryapplications. Mitochondria stained with the dye appear yellow-greenthrough a fluorescein longpass optical filter and red through atetramethylrhodamine longpass optical filter. Unlike the lipophilicrhodamine and carbocyanine dyes, rhodamine 123 does not stain theendoplasmic reticulum. Rhodamine-123 has been used with a variety ofcell types including nerve cells, bacteria, plant cells and spermatozoa,and has also been used to study apoptosis, axoplasmic transport ofmitochondria, bacterial viability and vitality, mitochondrial enzymaticactivities, transmembrane potential and other membrane activities,multidrug resistance, mycobacterial drug susceptibility and oocytematuration. Derivatives of rhodamine-123 such as TMRE have beendeveloped that are more permeable and have less hydrogen-bondinginteraction with anionic sites in the mitochondrial inner membrane andmatrix.

There continues to exist a need for novel antimicrobial agents usefulfor treating a variety of infections and for methods of identifying suchnovel compounds. Such methods ideally would provide for rapid and highlyselective identification of compounds that may be structurally distinctfrom the major conventional antimicrobial agents.

SUMMARY OF THE INVENTION

The present invention generally provides methods for identifyingantimicrobial compounds (including, for example, antifungal orantibacterial compounds) based on the discovery that a class ofantimicrobial agents based on or derived frombactericidal/permeability-increasing protein (BPI) generates uniqueeffects on fungal and bacterial cells as revealed by treatment with acyanine membrane potential indicator dye, DiOC₆(3). When BPI-derivedpeptide compounds are employed as antifungal agents, their effects arecharacterized by localization of the cyanine dye to mitochondria withincreasing accumulation of this dye in a peptide concentration-dependentmanner, and with retention of the dye notwithstanding an onset of lossor reduction of fungal cell viability at the same peptide concentration.The dye appears to be retained even after cell death has occurred (asconfirmed, e.g., by a negative 24-hour growth culture or by use of otherviability indicators, such as propidium iodide). Similarly, when rBPI₂₁and BPI-derived peptide compounds are employed as antibacterial agents,their effects are also characterized by increasing accumulation of thiscyanine dye in a peptide concentration-dependent manner, with retentionof the dye notwithstanding an onset of loss or reduction of bacterialviability at the same peptide concentration.

Novel antimicrobial agents may be rapidly and selectively identified byscreening test compounds for replication of the characteristic increasein dye fluorescence intensity produced by BPI protein products withcontinued retention of dye notwithstanding loss (i.e., reduction) ofviability within the tested target cell population. Sources of testcompounds include, for example, libraries (including combinatoriallibraries) of inorganic and organic compounds (for example, bacterial,fungal, mammalian, insect or plant products, peptides, peptidomimeticsand organomimetics). Presently preferred standard BPI-derivedantimicrobial peptides that are known to produce this characteristicpattern include XMP.391 (SEQ ID NO: 1) and XMP.445 (SEQ ID NO:2). Thisaspect of the invention thus contemplates a method of identifying anantimicrobial agent, particularly an antifungal compound, comprising thesteps of: (a) contacting a target cell (e.g., a fungal cell or abacterial cell) with a test compound and with a membrane potentialindicator dye, and (b) detecting an increasing accumulation of this dyeand retention of this dye despite loss or reduction of target cellviability. Presently preferred membrane potential indicator dyes areDiOC₆(3), JC-1, rhodamine-123 and MitoTracker Red CM-H₂Xros [M-7513,Molecular Probes, Inc., Eugene, Oreg.]. The concurrent loss or reductionof target cell viability may be confirmed by routine culture or throughuse of viability dyes, such as propidium iodide.

It is further contemplated that screening methods according to thepresent invention may involve multiple further stages of screening,including selection of test compounds that have a differential effect ontarget cells in comparison to non-target cells (e.g., a reduced effecton mammalian cells relative to fungal cells). This aspect of theinvention provides a further screening assay involving (a) contacting amammalian cell with the test compound and with the membrane potentialindicator dye, and (b) detecting no substantial increase in dyefluorescence intensity. Optionally, compounds may be screened forselectivity for one type of microbial cell, e.g., selectivity forbacterial vs. fungal cells or vice versa.

Test compounds may be alternatively or additionally assayed for abilityto kill or inhibit growth of target cells (e.g., fungal cells orbacteria) in vitro using any assays known in the art, including broth orradial diffusion assays. Suitable compounds may have a 2-fold, 10-fold,50-fold, 100-fold, or greater separation (selectivity) betweenantimicrobial activity and mammalian cell activity. The most desirablecompounds will preferably have a 50-fold or greater separation betweenantimicrobial activity and mammalian cell activity as quantified bydifferential effects on dye fluorescence intensity.

The in vivo antimicrobial activity of test compounds may also be assayedin any animal models of infection known to those skilled in the art.Such assays include those for in vitro and in vivo oral availability andthose for in vivo oral activity as evidenced by activity whenadministered orally in a comparative survival study.

Another aspect of the invention provides kits for use in conducting thescreening methods of the present invention. Such kits may optionallyinclude (a) a membrane potential indicator and (b) a BPI-derivedantimicrobial peptide or other BPI protein product suitable for use as astandard (positive control) against which the test compound may becompared.

The present invention also provides novel antimicrobial compoundsidentified by the screening methods of the present invention.

Yet a further aspect of the invention contemplates the treatment ofinfections, including fungal and bacterial infections, using compoundsidentified by the screening methods of the present invention thatexhibit the above-described characteristic pattern, other than compoundsknown in the art (including BPI protein products such as BPI-derivedpeptides).

Numerous additional aspects and advantages of the invention will becomeapparent to those skilled in the art upon consideration of the followingdetailed description of the invention which describes presently preparedembodiments thereof

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the effect of XMP.391 and XMP.445 on fungal and mammaliancells treated with cyanine dye DiOC₆(3).

FIG. 2 depicts the effect of XNP.391 and XMP.445 on fungal cells treatedwith propidium iodide and the effect of XMP.445 on mammalian cellstreated with propidium iodide.

FIG. 3 depicts the effect of miconazole on fungal and mammalian cellstreated with DiOC₆(3) and on mammalian cells treated with propidiumiodide.

FIG. 4 depicts the effect of XMP.391 on fungal cells treated with avariety of membrane potential indicator dyes.

FIG. 5 depicts the effect of rBPI₂₁, on E. coli J5 cells treated withcyanine dye DiOC₆(3) and propidium iodide and on E. coli O111 cellstreated with DiOC₆(3).

FIG. 6 depicts the effect of XMP.365 on E. coli cells treated withcyanine dye DiOC₆(3) and propidium iodide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally provides methods for identifyingantimicrobial compounds that mimic the unique effects of BPI proteinproducts, particularly BPI-derived antimicrobial peptides, on cellstreated with a membrane potential indicator dye. This unique“fingerprint” manifests as a dose-dependent increase and accumulation ofthe membrane potential indicator dye by the target cells (as measured,for example, by an increase in dye fluorescence intensity) and retentionof the dye despite the onset of loss or reduction of target cellviability. The invention is based on the discovery that antimicrobialagents based on or derived from bactericidal/permeability-increasingprotein (BPI) display unexpectedly unique effects on fungal cells andbacteria treated with a cyanine membrane potential indicator dye, suchas DiOC₆(3). A characteristic pattern of peptide concentration-dependentdye accumulation in target cells with retention of the dye in the targetcells at a time or peptide concentration when other indicators establishthat the target cells have lost viability provides an unexpected“fingerprint.” Dying target cells would not be expected to display anapparent increase in mitochondrial membrane potential and would not beexpected to retain a membrane potential indicator dye.

Any membrane potential indicator dyes that provide the above-describedunique “fingerprint” of BPI protein products, including any of the dyesnamed above (e.g., cyanine-, oxonol- or rhodamine-based dyes), may beused in the methods and kits of the present invention. For testingactivity of compounds against eukaryotic cells such as fungal cells,preferred dyes are dyes that localize to mitochondria. For example,Haugland, Handbook of Fluorescent Probes and Research Chemicals, Spence,ed., Molecular Probes, Inc., Eugene, Oreg. (1996) lists a number ofmitochondrial staining fluorescent dyes, including rhodamine 123 andrhodamine derivatives such as TMRM and TMRE, MitoTracker® OrangeCM-H₂TMRos and Red CM-H₂Xros dyes [M-7511 and M-7513, Molecular Probes,Inc., Eugene, Oreg.]; carbocyanine dyes such as DiOC₆(3), DiOC₂(5),DiOC₇(3), DiSC₂(5), DiOC₃(5) and DiOC₅(3); styryl dyes such as DASPMI(4Di-1-ASP and 2-Di-1-ASP) and DASPEI; JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide) and its analogs such as TDBC-3 and TDBC-4. Presently preferredis cyanine dye DiOC₆(3).

Any BPI protein product which displays the above-describedcharacteristic pattern of increase in dye fluorescence intensity may beused as a standard against which the test compound may be compared.Presently preferred are rBPI₂₁ and BPI-derived peptides, includingdomain III-derived peptides such as XMP.391 (SEQ ID NO: 1) [thestructure of which is described in Table 1 of U.S. Pat. No. 5,858,974and corresponding International Publication No. WO 97/04008(PCT/US96/03845), both of which are incorporated by reference herein]and XMP.445 (SEQ ID NO:2) [the structure of which is described inco-owned, U.S. Provisional Application Serial No. 60/101,958 filed Sep.25, 1998 and No. 60/109,896 filed Nov. 25, 1998, and co-owned,concurrently filed U.S. application Ser. No. 09/406,243, all of whichare incorporated by reference herein]. Procedures for the preparationand purification of BPI-derived peptides are described in, for example,U.S. Pat. Nos. 5,858,974, 5,733,872 and 5,652,332, incorporated hereinby reference.

As used herein, “BPI protein product” includes naturally andrecombinantly produced BPI protein; natural, synthetic, and recombinantbiologically active polypeptide fragments of BPI protein; biologicallyactive polypeptide variants of BPI protein or fragments thereof,including hybrid fusion proteins and dimers; biologically activepolypeptide analogs of BPI protein or fragments or variants thereof,including cysteine-substituted analogs; and BPI-derived peptides; all ofwhich are described in more detail in U.S. Pat. No. 5,627,153 andcorresponding International Publication No. WO 95/19179(PCT/US95/00498), both of which are incorporated herein by reference.

Test compounds may be assayed on any microbial organism, including thoseinvolved in pathogenic infection. Fungal species include, e.g., Candida(including C. albicans, C. tropicalis, C. parapsilosis, C. stellatoidea,C. krusei, C. parakrusei, C. lusitanae, C. pseudotropicalis, C.guilliermondi and C. glabrata), Aspergillus (including A. fumigatus, A.flavus, A. niger, A. nidulans, A. terreus, A. sydowi, A. flavatus, andA. glaucus), Cryptococcus, Histoplasma, Coccidioides, Paracoccidioides,Blastomyces, Basidiobolus, Conidiobolus, Rhizopus, Rhizomucor, Mucor,Absidia, Mortierella, Cunninghamella, Saksenaea, Pseudallescheria,Sporotrichosis, Fusarium, Trichophyton, Trichosporon, Microsporum,Epidermophyton, Scytalidium, Malassezia, Actinomycetes, Sporothrix,Penicillium, Saccharomyces and Pneumocystis. Gram-negative bacterialspecies that may be tested include Acidarinococcus, Acinetobacter,Aeromonas, Alcaligenes, Bacteroides, Bordetelia, Branhamella, Brucella,Calymmatobacterium, Campylobacter, Cardiobacterium, Chromobacterium,Citrobacter, Edwardsiella, Enterobacter, Escherichia, Flavobacterium,Francisella, Fusobacterium, Haemophilus, Klebsiella, Legionella,Moraxelia, Morganella, Neisseria, Pasturella, Plesiomonas, Proteus,Providencia, Pseudomonas, Salmonella, Serratia, Shigella,Streptobacillus, Veillonella, Vibrio, and Yersinia species; whilegram-positive bacterial species that may be tested includeStaphylococcus, Streptococcus, Micrococcus, Peptococcus,Peptostreptococcus, Enterococcus, Bacillus, Clostridium, Lactobacillus,Listeria, Erysipelothrix, Propionibacterium, Eubacterium, andCorynebacterium species. Protozoa include Plasmodia, Toxoplasma,Leishmania, Trypanosoma, Acanthamoeba, Nagleria, and Pneumocystisspecies.

Sources for test compounds to be screened include (1) inorganic andorganic chemical libraries, (2) natural product libraries, and (3)combinatorial libraries comprised of either random or mimetic peptides,oligonucleotides or organic molecules. Chemical libraries may be readilysynthesized or purchased from a number of commercial sources, and mayinclude structural analogs of known compounds or compounds that areidentified as “hits” or “leads” via natural product screening. Thesources of natural product libraries are collections of microorganisms(including bacteria and fungi), animals, plants or other vegetation,insects, including Arachnid species, or marine organisms, and librariesof mixtures for screening may be created by: (1) fermentation andextraction of broths from soil, plant or marine microorganisms or (2)extraction of the organisms themselves. Natural product librariesinclude polyketides, non-ribosomal peptides, and variants (non-naturallyoccurring) variants thereof For a review, see Science 282:63-68 (1998).Combinatorial libraries are composed of large numbers of peptides,oligonucleotides or organic compounds and can be readily prepared bytraditional automated synthesis methods, PCR, cloning or proprietarysynthetic methods. Of particular interest are peptide andoligonucleotide combinatorial libraries. Still other libraries ofinterest include peptide, protein, peptidomimetic, multiparallelsynthetic collection, recombinatorial, and polypeptide libraries. For areview of combinatorial chemistry and libraries created therefrom, seeMyers, Curr. Opin. Biotechnol. 8:701-707 (1997). For reviews andexamples of peptidomimetic libraries, see Al-Obeidi et al., Mol.Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol,1(1):114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996)(alkylated dipeptides). A variety of companies have constructed chemicallibraries and provide their use for screening, including for example,3-Dimensional Pharmaceuticals, Exton, Pa.; Agouron Pharmaceutical, LaJolla, Calif.; Alanex Corp., San Diego, Calif.; Ariad Pharmaceuticals,Cambridge, Mass.; ArQule, Inc., Medford, Mass.; Arris Pharmaceutical, S.San Francisco, Calif.; Axys, S. San Francisco, Calif.; BiocrystPharmaceuticals, Birmingham, Ala.; Cadus Pharmaceuticals, Tarrytown,N.Y.; Cambridge Combinatorial, Cambridge, UK; ChemGenics, Cambridge,Mass.; CombiChem, San Diego, Calif.; Corvas International, San Diego,Calif.; Cubist Pharmaceuticals, Cambridge, Mass.; Darwin Molecular,Bothell, Wash.; Houghten Pharmaceuticals, San Diego, Calif.; Hybridon,Cambridge, Mass.; Isis Pharmaceuticals, Carlsbad, Calif.; Ixsys, SanDiego, Calif.; Molecumetics, Bellevue, Wash.; Peptide Therapeutics,Cambridge, UK; Pharmacopia, Princeton, N.J.; SUGEN, Redwood City,Calif.; Telik, Inc., S. San Francisco, Calif.; and Tripos, Inc., St.Louis, Mo.

Preferably the compounds that are preliminarily identified by thismethod are then assayed by conventional methods known in the art for theability to kill or inhibit growth/replication of whole target cels invitro. Such assays may include the steps of contacting test compoundswith whole target cells and measuring viability or proliferation of thetarget cells. Any assays known in the art may be used, including thosedescribed in Examples 2 and 3 of U.S. Pat. No. 5,858,974.

Some compounds may be more suitable for in vitro use, including, forexample, use as a preservative or decontaminant for fluids and surfaces,or use to sterilize surgical and other medical equipment and implantabledevices, either ex vivo or in situ, including prosthetic joints andindwelling invasive devices such as intravenous lines and catheterswhich are often foci of infection, or use in the preparation of growthmedia for non-target cells.

Ideally, the most desirable compounds for in vivo administration tomammals will have a differential effect on target and mammalian cells,i.e., if the compound does adversely affect mammalian cells, a higherconcentration of the compound would be required to affect the mammaliancells in comparison to target cells, thereby providing a therapeuticwindow of suitable concentrations for administering the compound withoutundesirable toxic effects. The relative effect on target and mammaliancells may be determined using any in vitro assays known in the art,including by contacting mammalian cells with the same test compound andthe same membrane potential indicator dye utilized for the initialantimicrobial screen, and selecting compounds that do not produce asubstantial change in dye uptake.

The potential antimicrobial compounds may also be evaluated for theireffect in any model of infection, including any in vivo model, known inthe art. Exemplary animal models of fungal infection are described inExample 4 of U.S. Pat. No. 5,858,974 and may be modified for any fungalspecies (including Candida, Aspergillus and Fusarium). Other microbialinfection models are known in the art. The most desirable compounds arecapable of preventing the establishment of an infection or reversing theoutcome of an infection once it is established without excessivetoxicity.

The use of antimicrobial compounds identified by the screening methodsof the present invention is contemplated for the treatment of subjectssuffering from microbial infection, especially mammalian subjects suchas humans, but also including farm animals such as cows, sheep, pigs,horses, goats and poultry (e.g., chickens, turkeys, ducks and geese),companion animals such as dogs and cats, exotic and/or zoo animals, andlaboratory animals including mice, rats, rabbits, guinea pigs, andhamsters. Treatment of infection of plants is also contemplated.“Treatment” as used herein encompasses both prophylactic and therapeutictreatment, and may be accompanied by concurrent administration of otherantimicrobial agents, including any of the agents discussed herein.

Therapeutic compositions may be administered systemically or topically.Systemic routes of administration include oral, intravenous,intramuscular or subcutaneous injection (including into a depot forlong-term release), intraocular and retrobulbar, intrathecal,intraperitoneal (e.g. by intraperitoneal lavage), intrapulmonary (usingpowdered drug, or an aerosolized or nebulized drug solution), ortransdermal. Suitable dosages include doses ranging from 1 μg/kg to 100mg/kg per day and doses ranging from 0.1 mg/kg to 20 mg/kg per day. Forpolypeptide therapeutics that are amenable to administration via genetherapy, methods of delivering suitable genes to a subject (includingplants and animals) are contemplated. Those skilled in the art canreadily optimize effective dosages and administration regimens fortherapeutic compositions as determined by good medical practice and theclinical condition of the individual subject.

“Concurrent administration,” or “co-administration,” as used hereinincludes administration of one or more agents, in conjunction orcombination, together, or before or after each other. The agents may beadministered by the same or by different routes. If administered via thesame route, the agents may be given simultaneously or sequentially, aslong as they are given in a manner sufficient to allow all agents toachieve effective concentrations at the site of action.

Known antifungal agents include polyene derivatives, such asamphotericin B (including lipid or liposomal formulations thereof) andthe structurally related compounds nystatin and pimaricin; flucytosine(5-fluorocytosine); azole derivatives (including ketoconazole,clotrimazole, miconazole, econazole, butoconazole, oxiconazole,sulconazole, tioconazole, terconazole, fluconazole, itraconazole,voriconazole [Pfizer] and SCH56592 [Schering-Plough]);allylamines-thiocarbamates (including tolnaftate, naftifine andterbinafine); griseofulvin; ciclopirox; haloprogin; echinocandins(including MK-0991 [Merck]); and nikkomycins. Recently discovered asantifungal agents are a class of products related tobactericidal/permeability-increasing protein (BPI), described in U.S.Pat. Nos. 5,627,153, 5,858,974, 5,652,332, 5,763,567 and 5,733,872, thedisclosures of all of which are incorporated herein by reference.

The polyene derivatives, which include amphotericin B and thestructurally related compounds nystatin and pimaricin, arebroad-spectrum antifungals that bind to ergosterol, a component offungal cell membranes, and thereby disrupt the membranes. Amphotericin Bis usually effective for systemic mycoses, but its administration islimited by toxic effects that include fever and kidney damage, and otheraccompanying side effects such as anemia, low blood pressure, headache,nausea, vomiting and phlebitis. The unrelated antifungal agentflucytosine (5-fluorocytosine), an orally absorbed drug, is frequentlyused as an adjunct to amphotericin B treatment for some forms ofcandidiasis and cryptococcal meningitis. Its adverse effects includebone marrow depression with leukopenia and thrombocytopenia.

The azole derivatives impair synthesis of ergosterol and lead toaccumulation of metabolites that disrupt the function of fungalmembrane-bound enzyme systems (e.g., cytochrome P450) and inhibit fungalgrowth. This group of agents includes ketoconazole, clotrimazole,miconazole, econazole, butoconazole, oxiconazole, sulconazole,tioconazole, terconazole, fluconazole and itraconazole. Significantinhibition of mammalian P450 results in significant drug interactions.Some of these agents may be administered to treat systemic mycoses.Ketoconazole, an orally administered imidazole, is used to treatnonmeningeal blastomycosis, histoplasmosis, coccidioidomycosis andparacoccidioidomycosis in non-immunocompromised patients, and is alsouseful for oral and esophageal candidiasis. Adverse effects include raredrug-induced hepatitis; ketoconazole is also contraindicated inpregnancy. Itraconazole appears to have fewer side effects thanketoconazole and is used for most of the same indications. Fluconazolealso has fewer side effects than ketoconazole that is used for oral andesophageal candidiasis and cryptococcal meningitis. Miconazole is aparenteral imidazole with efficacy in coccidioidomycosis and severalother mycoses, but has side effects including hyperlipidemia andhyponatremia.

The allylamines-thiocarbamates are generally used to treat skininfections. This group includes tolnaftate, naftifine and terbinafine.Another antifungal agent is griseofulvin, a fungistatic agent which isadministered orally for fungal infections of skin, hair or nails that donot respond to topical treatment. Other topical agents includeciclopirox and haloprogin. [Chapter 49 in Goodman and Gilman, ThePharmacological Basis of Therapeutics, 9th ed., McGraw-Hill, New York(1996), pages 1175-1190.]

BPI protein products, a class of products related tobactericidal/permeability-increasing protein (BPI), are described inU.S. Pat. No. 5,627,153 and corresponding International Publication No.WO 95/19179 (PCT/US95/00498), all of which are incorporated by referenceherein, to have antifungal activity. BPI-derived peptides withantifungal activity are described in U.S. Pat. No. 5,858,974, which isin turn a continuation-in-part of U.S. application Ser. No. 08/504,841filed Jul. 20, 1994 and corresponding International Publication Nos. WO96/08509 (PCT/US95/09262) and WO 97/04008 (PCT/US96/03845), all of whichare incorporated by reference herein. Other peptides with antifungalactivity are described in U.S. Pat. No. 5,652,332 [corresponding toInternational Publication No. WO 95/19372 (PCT/US94/10427)], and in U.S.Pat. Nos. 5,763,567 and 5,733,872 [corresponding to InternationalPublication No. WO 94/20532 (PCT/US94/02465)], which is acontinuation-in-part of U.S. patent application Ser. No. 08/183,222filed Jan. 14, 1994, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/093,202 filed Jul. 15, 1993 [corresponding toInternational Publication No. WO 94/20128 (PCT/US94/02401)], which is acontinuation-in-part of U.S. patent application Ser. No. 08/030,644filed Mar. 12, 1993, now U.S. Pat. No. 5,348,942, the disclosures of allof which are incorporated herein by reference.

Known antibacterial agents include antibiotics, which are naturalchemical substances of relatively low molecular weight produced byvarious species of microorganisms, such as bacteria (including Bacillusspecies), actinomycetes (including Streptomyces) and fungi, that inhibitgrowth of or destroy other microorganisms. Substances of similarstructure and mode of action may be synthesized chemically, or naturalcompounds may be modified to produce semi-synthetic antibiotics. Thesebiosynthetic and semi-synthetic derivatives are also effective asantibiotics. The major classes of antibiotics are (1) the β-lactams,including the penicllins, cephalosporins and monobactams; (2) theaminoglycosides, e.g., gentamicin, tobramycin, netilmycin, and amikacin;(3) the tetracyclines; (4) the sulfonamides and trimethoprim; (5) thefluoroquinolones, e.g., ciprofloxacin, norfloxacin, and ofloxacin; (6)vancomycin; (7) the macrolides, which include for example, erythromycin,azithromycin, and clarithromycin; and (8) other antibiotics, e.g., thepolymyxins, chloramphenicol and the lincosamides.

Antibiotics accomplish their anti-bacterial effect through severalmechanisms of action which can be generally grouped as follows: (1)agents acting on the bacterial cell wall such as bacitracin, thecephalosporins, cycloserine, fosfomycin, the penicillins, ristocetin,and vancomycin; (2) agents affecting the cell membrane or exerting adetergent effect, such as colistin, novobiocin and polymyxins; (3)agents affecting cellular mechanisms of replication, informationtransfer, and protein synthesis by their effects on ribosomes, e.g., theaminoglycosides, the tetracyclines, chloramphenicol, clindamycin,cycloheximide, fucidin, lincomycin, puromycin, rifampicin, otherstreptomycins, and the macrolide antibiotics such as erythromycin andoleandomycin; (4) agents affecting nucleic acid metabolism, e.g., thefluoroquinolones, actinomycin, ethambutol, 5-fluorocytosine,griseofulvin, rifamycins; and (5) drugs affecting intermediarymetabolism, such as the sulfonamides, trimethoprim, and thetuberculostatic agents isoniazid and para-aminosalicylic acid. Someagents may have more than one primary mechanism of action, especially athigh concentrations. In addition, secondary changes in the structure ormetabolism of the bacterial cell often occur after the primary effect ofthe antimicrobial drug.

The penicillins have a characteristic double-ring system composed of aβ-lactam ring, which provides the antibacterial activity, and athiazolidene ring. The penicillins are differentiated by a single sidechain that is unique for each penicillin. The compounds are bactericidaland act by inhibiting bacterial transpeptidase, an enzyme involved insynthesis of the bacterial cell wall. Because of their mechanism ofaction, penicillins are generally active against growing, but notresting, cells. Penicillins, especially penicillin G, have largelygram-positive activity; the relative insensitivity of gram-negative rodsto penicillin G and several other penicillins is probably due to thepermeability barrier of the outer membrane of gram-negative bacteria.Ampicillin, carbenicillin, ticarcillin, and some other penicillins areactive against gram-negative bacteria because they can pass through thisouter membrane. Penicillins have relatively few adverse effects, themost important of which are the hypersensitivity (allergic) reactions.These compounds are widely distributed in the body, but do not entercells and do not usually accumulate in CSF.

Bacterial resistance to the penicillins is by production of the enzymeβ-lactmase, which catalyzes hydrolysis of the β-lactam ring. Thepercentage of bacteria resistant to penicillin has risen to about 80%.Several penicillins, including methicillin, oxacillin, cloxacillin,dicloxacillin and nafcillin, are not affected by the β-lactamase ofstaphylococci. These antibiotics are useful against mostβ-lactamase-producing species of Staphylococcus. However, a small numberof species are resistant even to these penicilins. Some penicillins,amoxicillin and ticarcillin, are marketed in combination with clavulanicacid, which is a β-lactamase inhibitor that covalently binds to theenzyme and prevents it from hydrolyzing the antibiotics. Anotherinhibitor, sulbactam, is marketed in combination with ampicillin.

The cephalosporins are characterized by a β-lactam ring, like thepenicillins, but have an adjacent dihydrothiazine ring instead of athiazolidene ring. For convenience, these compounds are generallyclassified by generations. The first generation includes cephalothin,cephapirin, cefazolin, cephalexin, cephradine and cefadroxil. Thesedrugs generally have excellent gram-positive activity except forenterococci and methicillin-resistant staphylococci, and have onlymodest gram-negative coverage. The second generation includescefamandole, cefoxitin, ceforanide, cefuroxime, cefuroxime axetil,cefaclor, cefonicid and cefotetan. This generation generally loses somegram-positive activity by weight and gains limited gram-negativecoverage. The third generation includes cefotaxime, moxalactam,ceftizoxime, ceftriaxone, cefoperazone and ceftazidime. These compoundsgenerally sacrifice further gram-positive activity by weight but gainsubstantial gram-negative coverage against Enterobacter and sometimesare active against Pseudomonas. The cephalosporins bind topenicillin-binding proteins with varying affinity. Once binding occurs,protein synthesis is inhibited. Cephalosporins are usually welltolerated; adverse effects include hypersensitivity reactions andgastrointestinal effects. Cephalosporins may interact with nephrotoxicdrugs, particularly aminoglycosides, to increase toxicity. Resistance tocephalosporins is mediated by several mechanisms, including productionof β-lactamase, although some strains that do not produce β-lactamaseare nevertheless resistant.

Imipenem is a N-formimidoyl derivative of the mold product thienamycin.It contains a β-lactam ring and somewhat resembles penicillin except fordifferences in the second ring. It has activity against bothgram-positive and gram-negative organisms and is resistant to mostP-lactamases, although not those from Pseudomonas. It is marketed incombination with cilastin, a compound that inhibits inactivation ofimipenem in the kidney by renal dihydropeptidase I enzyme. Cilastinincreases the concentration of imipenem in urine, although not in blood.

Aztreonam is the first of a new group of antibiotics referred to as themonobactams. These agents have a P-lactam ring but lack the second ringcharacteristic of the penicillins and cephalosporins. It acts by bindingto penicillin-binding proteins, and produces long, filamentous bacterialshapes that eventually lyse. Aztreonam is active only against aerobicgram-negative bacteria, is susceptible to inactivation by someβ-lactamases, and has few adverse effects.

The aminoglycosides contain amino sugars linked to an aminocyclitol ringby glycosidic bonds. They have similar mechanisms of action andproperties, but differ somewhat in spectrum of action, toxicity, andsusceptibility to bacterial resistance. The compounds are bactericidal,with activity against both gram-positive and gram-negative organisms,and act by binding to proteins on the 30S ribosome of bacteria andinhibiting protein synthesis. The aminoglycosides also bind to isolatedLPS and have a very weak outer membrane permeabiUzng effect. [Taber etal., Microbiological Reviews 53: 439-457 (1987)); Kadurugamuwa et al.,Antimicrobial Agents and Chemotherapy, 37: 715-721 (1993); Vaara,Microbiological Reviews 56: 395-411 (1992)]. This class of antibioticsincludes amikacin, gentamicin, kanamycin, neomycin, netilmycin,paromomycin and tobramycin. The aminoglycosides are usually reserved formore serious infections because of severe adverse effects includingototoxicity and nephrotoxicity. There is a narrow therapeutic windowbetween the concentration required to produce a therapeutic effect,e.g., 8 μg/ml for gentamicin, and the concentration that produces atoxic effect, e.g., 12 μg/ml for gentamicin. Neomycin in particular ishighly toxic and is never administered parentemuly.

Tetracyclines have a common four-ring structure and are closelycongeneric derivatives of the polycyclic naphthacenecarboxamide. Thecompounds are bacteriostatic, and inhibit protein synthesis by bindingto the 30S subunit of microbial ribosomes and interfering withattachment of aminoacyl tRNA. The compounds have some activity againstboth gram-positive and gram-negative bacteria; however, their use islimited because many species are now relatively resistant. Adverseeffects include gastrointestinal effects, hepatotoxicity with largedoses, and nephrotoxicity in some patients. This antibiotic classincludes tetracycline, chlortetracycline, demeclocycline, doxycycline,methacycline, minocycline and oxytetracycline.

The sulfonamides are derivatives of sulfanilamide, a compound similar instructure to para-aminobenzoic acid (PABA), which is an essentialprecursor for bacterial synthesis of folic acid. The compounds aregenerally bacteriostatic, and act by competitively inhibitingincorporation of PABA into tetrahydrofolic acid, which is a requiredcofactor in the synthesis of thymidines, purines and DNA. Sulfonamideshave a wide range of activity against gram-positive and gram-negativebacteria, but their usefulness has diminished with increasingly highprevalence of bacterial resistance. The sulfonamide class of antibioticsincludes sulfacytine, sulfadiazine, sulfamethizole, sulfisoxazole,sulfamethoxazole, sulfabenzamide and sulfacetamide. Adverse effectsinclude hypersensitivity reactions and occasional hematologicaltoxicity.

Trimethoprim is an inhibitor of the dihydrofolate reductase enzyme,which converts dihydrofolic to tetrahydrofolic acid, a required factorfor DNA synthesis. Adverse effects include gastrointestinal distress andrare hematological toxicity. Trimethoprim is also available incombination with sulfamethoxazole (also known as co-trimoxazole). Thecombination is usually bactericidal, although each agent singly isusually bacteriostatic. The combination is the drug of choice forSalmonella infections, some Shigella infections, E. coli traveler'sdiarrhea and Pneumocystis carinii pneumonia.

The fluoroquinolones and quinolones are derivatives of nalidixic acid, anaphthyridine derivative. These compounds are bactericidal, and impairDNA replication, transcription and repair by binding to the DNA andinterfering with DNA gyrase, an enzyme which catalyzes negativesupercolling of DNA. The fluoroquinolones, which include norfloxacin,ciprofloxacin, and ofloxacin, and the quinolones, which includecinoxacin, have a broad spectrum of antimicrobial activity againstgram-negative and gram-positive organisms. These compounds distributewidely through extravascular tissue sites, have a long serum half-life,and present few adverse effects. Because of their effect on DNA, thedrugs are contraindicated in pregnant patients and in children whoseskeletal growth is incomplete.

Vancomycin is a glycopeptide, with a molecular weight of about 1500,produced by a fungus. It is primarily active against gram-positivebacteria. The drug inhibits one of the final steps in synthesis of thebacterial cell wall, and is thus effective only against growingorganisms. It is used to treat serious infections due to gram-positivecocci when penicillin G is not useful because of bacterial resistance orpatient allergies. Vancomycin has two major adverse effects, ototoxicityand nephrotoxicity. These toxicities can be potentiated by concurrentadministration of another drug with the same adverse effect, such as anaminoglycoside.

The macrolides are bacteriostatic and act by binding to the 50S subunitof 70S ribosomes, resulting in inhibition of protein synthesis. Theyhave a broad spectrum of activity against gram-positive andgram-negative bacteria and may be bacteriostatic or bactericidal,depending on the concentration achieved at sites of infection. Thecompounds distribute widely in body fluids. Adverse effects includegastrointestinal distress and rare hypersensitivity reactions. The mostcommon macrolide used is erythromycin, but the class includes othercompounds such as clarithromycin and azithromycin.

The polymyxins are a group of closely related antibiotic substancesproduced by strains of Bacillus polymyxa. These drugs, which arecationic detergents, are relatively simple, basic peptides withmolecular weights of about 1000. Their antimicrobial activity isrestricted to gram-negative bacteria. They interact strongly withphospholipids and act by penetrating into and disrupting the structureof cell membranes. Polymyxin B also binds to the lipid A portion ofendotoxin and neutralizes the toxic effects of this molecule. PolymyxinB has severe adverse effects, including nephrotoxicity andneurotoxicity, and should not be administered concurrently with othernephrotoxic or neurotoxic drugs. The drug thus has limited use as atherapeutic agent because of high systemic toxicity, but may be used forsevere infections, such as Pseudomonas aeruginosa meningitis, thatrespond poorly to other antibiotics.

Chloramphenicol inhibits protein synthesis by binding to the 50Sribosomal subunit and preventing binding of aminoacyl tRNA. It has afairly wide spectrum of antimicrobial activity, but is only reserved forserious infections, such as meningitis, typhus, typhoid fever, and RockyMountain spotted fever, because of its severe and fatal adversehematological effects. It is primarily bacteriostatic, although it maybe bactericidal to certain species.

Lincomycin and clindamycin are lincosamide antimicrobials. They consistof an amino acid linked to an amino sugar. Both inhibit proteinsynthesis by binding to the 50S ribosomal subunit. They compete witherythromycin and chloramphenicol for the same binding site but in anoverlapping fashion. They may be bacteriostatic or bactericidal,depending on relative concentration and susceptibility. Gastrointestinaldistress is the most common side effect. Other adverse reactions includecutaneous hypersensitivity, transient hematological abnormalities, andminor elevations of hepatic enzymes. Clindamycin is often the drug ofchoice for infections caused by anaerobic bacteria or mixedaerobic/anaerobic infections, and can also be used for susceptibleaerobic gram-positive cocci.

Some drugs, e.g. aminoglycosides, have a small therapeutic window. Forexample, 2 to 4 μg/ml of gentamicin or tobramycin may be required forinhibition of bacterial growth, but peak concentrations in plasma above6 to 10 μg/ml may result in ototoxicity or nephrotoxicity. These agentsare more difficult to administer because the ratio of toxic totherapeutic concentrations is very low. Antimicrobial agents that havetoxic effects on the kidneys and that are also eliminated primarily bythe kidneys, such as the aminoglycoside or vancomycin, requireparticular caution because reduced elimination can lead to increasedplasma concentrations, which in turn may cause increased toxicity. Dosesof antimicrobial agents that are eliminated by the kidneys must bereduced in patients with impaired renal function. Similarly, dosages ofdrugs that are metabolized or excreted by the liver, such aserythromycin, chloramphenicol, or clindamycin, must be reduced inpatients with decreased hepatic function.

Bacteria acquire resistance to antibiotics through several mechanisms:(1) production of enzymes that destroy or inactivate the antibiotic[Davies, Science, 264:375-381 (1994)]; (2) synthesis of new or alteredtarget sites on or within the cell that are not recognized by theantibiotic [Spratt, Science, 264:388-393 (1994)]; (3) low permeabilityto antibiotics, which can be reduced even further by altering cell wallproteins, thus restricting access of antibiotics to the bacterialcytoplasmic machinery; (4) reduced intracellular transport of the drug;and (5) increased removal of antibiotics from the cell viamembrane-associated pumps [Nikaido, Science, 264:382-387 (1994)].

The susceptibility of a bacterial species to an antibiotic is generallydetermined by two microbiological methods. A rapid but crude procedureuses commercially available filter paper disks that have beenimpregnated with a specific quantity of the antibiotic drug. These disksare placed on the surface of agar plates that have been streaked with aculture of the organism being tested, and the plates are observed forzones of growth inhibition. A more accurate technique, the brothdilution susceptibility test, involves preparing test tubes containingserial dilutions of the drug in liquid culture media, then inoculatingthe organism being tested into the tubes. The lowest concentration ofdrug that inhibits growth of the bacteria after a suitable period ofincubation is reported as the minimum inhibitory concentration.

The resistance or susceptibility of an organism to an antibiotic isdetermined on the basis of clinical outcome, i.e., whetheradministration of that antibiotic to a subject infected by that organismwill successfully cure the subject. While an organism may literally besusceptible to a high concentration of an antibiotic in vitro, theorganism may in fact be resistant to that antibiotic at physiologicallyrealistic concentrations. If the concentration of drug required toinhibit growth of or kill the organism is greater than the concentrationthat can safely be achieved without toxicity to the subject, themicroorganism is considered to be resistant to the antibiotic. Tofacilitate the identification of antibiotic resistance or susceptibilityusing in vitro test results, the National Committee for ClinicalLaboratory Standards (NCCLS) has formulated standards for antibioticsusceptibility that correlate clinical outcome to in vitrodeterminations of the minimum inhibitory concentration of antibiotic.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative examples. Example 1addresses the effect of two BPI-derived peptides, XMP.391 and XMP.445,on fungal cells and mammalian cells treated with the cyanine membranepotential indicator dye, DiOC₆(3). Example 2 addresses the concurrenteffect of these same BPI-derived peptides on fungal cells treated withpropidium iodide (PI), an indicator of cell viability. Example 3addresses in vitro oral absorption screening of BPI-derived peptides andExample 4 addresses in vivo oral absorption screening of BPI-derivedpeptides. Example 5 addresses the in vivo oral activity of XMP.445 in amouse survival efficacy study. Example 6 addresses the effect of theantifungal agent miconazole on fungal cells and mammalian cells treatedwith DiOC₆(3). Example 7 addresses the effect of BPI-derived peptides onfungal cells treated with the membrane potential indicator dyes JC-1,dihydrorhodamine 123, DiOC₆(3) and MitoTracker® Red CM-H₂Xros. Example 8shows localization of the accumulation of membrane potential indicatordye using confocal microscopy. Example 9 addresses the effect of a BPIprotein product on bacteria treated with DiOC₆(3).

EXAMPLE 1 Effect of XMP.391 and XMP.445 on Fungal and Mammalian CellsTreated with DiOC₆(3)

The relative effect of two BPI-derived peptides, XMP.391 and XMP.445, onfungal and mammalian cells treated with the cyanine membrane potentialindicator dye DiOC₆(3) (3,3′-dihexyloxacarbocyanine iodide) [MolecularProbes, Inc., Eugene, Oreg.], was assessed as follows.

A stock fungal cell suspension was prepared as follows. Fungi (Candidaalbicans strain SLU#1) were cultured on Sabouraud's dextrose agar plates[1 L water, 10 g neopeptone, 20 g dextrose, 15 g Bacto Agar]. One or twocolonies from the agar plate were inoculated into 5 mL of Sabouraud'sdextrose broth (SDB) [1 L water, 10 g neopeptone, 20 g dextrose] in asterile 10 mL polypropylene tube and incubated for about 18 hours at 30°C. After this incubation, 4 mL of the fungal culture was inoculated intoa flask containing 100 mL of SDB and incubated for about 5 hours oruntil log growth phase was reached. The culture was centrifuged at 3000rpm for 5 minutes (Sorvall RT6000B centrifuge), the supernatant wasdecanted and the pellets were resuspended in a total of 30 mL of SDB.Fungal cell concentration was determined either by absorbance at 570 nmor by diluting 1:10 with Trypan Blue and counting the number of fungalcells using a hemacytometer. The suspension was diluted with SDB toobtain a stock fungal cell suspension of approximately 1-2×10⁶ CFU/mL.

Stock 1 mg/mL solutions of peptides XMP.391 and XMP.445 were prepared insaline, and six 2-fold serial dilutions were prepared using phosphatebuffered saline (PBS). The fungal cell suspension was divided into 1 mLaliquots, and approximately 20 μL of each serial dilution of XMP.391 orXMP.445 peptide solution was added to an aliquot of fungal cellsuspension to obtain final peptide concentrations of 0.313, 0.625, 1.25,2.5, 5, 10 and 20 μg/mL in the aliquots. No peptide solution was addedto the auto-fluorescence and baseline controls. The aliquots wereincubated for 1 hour at 30° C. After the incubation, all aliquots werecentrifuged at 3000 rpm for 5 minutes (Sorvall RT6000B) and thesupernatant removed. The auto-fluorescence control aliquot wasresuspended in 1 mL of SDB; the remainder of the samples and thebaseline control were resuspended in 1 mL of 10 ng/mL DiOC₆(3)/SDB. Theauto-fluorescence control thus contained fungal cells in SDB alone(without XMP peptide and without dye), while the baseline controlcontained fungal cells with dye (but without XMP peptide). After a 20 to30 minute incubation in the dark at 30° C., the samples and controlswere centrifuged, the SDB was removed, and the pellets were resuspendedin 1 mL of PBS.

The samples were analyzed for fluorescence on the FACScan flowcytometer[Becton-Dickinson, Mountain View, Calif.] using the fl₁ detector at awavelength of 530 nm using the following parameters:

Amplifier Detector FSC 1.00-2.00 E00 SSC 1.00-2.00 200-300 FL1 (530 nm)Log 400-500 FL2 (585 nm) Log 400-500

The auto-fluorescence control was used for gating and fluorescencesensitivity was determined using the baseline control. The percentchange in fluorescent intensity, expressed as mean channels, wasdetermined by dividing the difference between the sample and baselinevalues by the baseline value: (sample fl₁−baseline fl₁)/baselinefl₁×100.

The same procedure was also carried out using mammalian Madin-Derbycanine kidney epithelial (MDCK) cells (ATCC Accession No. CCL34). TheMDCK cells were cultured until confluent in MEM media supplemented with10% fetal calf serum (FCS) and L-glutamine. The cells were harvested andresuspended to approximately 1-2×10⁶ cells/mL in RPMI media, treatedwith XMP.391 or XMP.445 at concentrations varying from 5 μg/mL to 100μg/mL, and incubated for 1 hour. The cells were then stained withDiOC₆(3) for 30 minutes and fluorescence intensity was determined in thesame manner as described above for C. albicans.

The results, displayed graphically in FIG. 1 as percent change influorescence mean channel from baseline vs. concentration of XMPpeptide, show that treatment of the C. albicans cells with XMP peptideand DiOC₆(3) dye resulted in a concentration-dependent increase influorescence intensity with increasing concentration of XMP peptide,despite a loss of fungal cell viability that occurs at a peptideconcentration of about 2 μg/mL for XMP.391 and about 4 μg/mL forXMP.445. In separate experiments, XMP.391 and XMP.445 were shown to haveminimum fungicidal concentration values of 2 μg/mL and 4 μg/mL,respectively, against this C. albicans SLU#1 strain.

Loss of fungal cell viability after XPM peptide treatment was confirmednot only by increased propidium iodide uptake as described in Example 2below but also by culture, which showed no detectable fungal cell growthafter the aliquots were incubated for 24 hours. Even after 24 hours, thealiquots were observed to retain their increased fluorescence intensity.

In contrast, treatment of the MDCK cells with the two XMP peptides andDiOC₆(3) dye resulted in no substantial increase in fluorescenceintensity even at 100-fold higher concentrations of XMP peptide. Theseresults demonstrate that peptides XMP.391 and XMP.445 show a selectiveeffect on fungal cells in comparison to mammalian cells.

EXAMPLE 2 Effect of XMP.391 and XMP.445 on Fungal and Mammalian CellsTreated with PI

The effect of two BPI-derived peptides, XMP.391 and XMP.445, on fungalcells (C. albicans SLU#1) treated with a viability dye, propidium iodide(PI) (Sigma, St. Louis, Mo.), and the effect of XMP.445 on mammalian MCKcells treated with PI, was assessed generally according to Example 1above, except that after the initial 1-hour incubation, samples andcontrols (including a baseline control and a positive control) wereresuspended in 1 mL of 10 μg/mL propidium iodide/Dulbecco's PBS ratherthan 10 ng/mL DiOC₆(3)/SDB and stained for 20 minutes in the dark atroom temperature. As in Example 1, the auto-fluorescence controlcontained fungal or mammalian cells in media alone (without XMP peptideand without PI), while the baseline control contained fungal ormammalian cells with PI (but without XMP peptide). Because it is aviability dye, PI is taken up only when cells are dying or dead; thefluorescent intensity of PI-treated samples should increase with thenumber of dead cells and should be maximal with 100% dead cells. Thus,for this experiment, a positive control was also prepared (containing100% dead cells) by resuspending cells in 1 mL of 70% ethanol during thelast 10 minutes of the initial 1-hour incubation; this positive controlthus contained 100% dead cells with PI (but without XMP peptide).Samples were analyzed for PI uptake on the FACScan using the fl₂detector at a wavelength of 585 nm. The auto-fluorescence sample wasused for gating, while the fluorescence sensitivity was determined bythe positive control and the PI uptake threshold was determined by thebaseline control. The percent change in fluorescent intensity in meanchannels was determined using the same equation used in Example 1, andthe percentage of cells with PI uptake (ie., dead cells) was calculated.The results are displayed graphically in FIG. 2 as percent of cells withPI uptake vs. concentration of peptide. These results, taken togetherwith FIG. 1, confirm that treatment with XMP.391 and XMP.445 resulted inincreasing accumulation of dye in fungal cells (as measured by increaseddye fluorescence intensity) and retention of dye despite reduction offungal cell viability or fungal cell death. These results also show thatXMP.445 did not substantially affect viability of mammalian cells, evenat a concentration of 50-fold or greater.

EXAMPLE 3 In Vitro Oral Absorption Screening

Various BPI-derived peptides were screened for oral absorption in invitro screening assays using CACO-2 and MDCK cells. Cultured monolayersof CACO-2 (Human colon carcinoma) [Audus, K. L., et al. Pharm. Res., 7:435-451(1990)] or Madin-Derby canine kidney epithelial (MDCK) cells(ATCC Accession No. CCL34) were grown upon collagen-coated,permeable-filter supports (Becton Dickenson, Mountainview, Calif.). Thecells were grown to confluency and allowed to differentiate. Theintegrity of the monolayers was determined by measuring thetransepithelial resistance. The cells were incubated with peptide on theapical side for 2.5 hours in MDCK screening or 4 hours for CACO-2screening. The transepithelial transport of the peptide was measured byquantitative HPLC analysis of the incubation media on the basolateralside of the cells. Radiolabelled mannitol and cortisone were used aspositive controls.

Intestinal absorption screening of peptides XMP.365, XMP.391 and XMP.445identified XMP.445 as a potential orally available compound.

EXAMPLE 4 In Vivo Oral Absorption Screening

Various BPI-derived peptides are screened for oral absorption in an invivo screening assay in which the peptides are administered by oralgavage to mice. Serum concentrations of the peptides are measured atvarious time intervals after administration by HPLC. Specifically,peptides are administered to mice at dosages of either 10 mg/kg bodyweight or 20 mg/kg body weight and the serum concentrations are measuredat intervals of 1 hour 4 hours and 24 hours after administration to themice. Peptide analysis indicates absorption after oral administrationand serum concentrations achieved.

EXAMPLE 5 Oral Activity of XMP.445

XMP.445 was tested for activity upon oral administration (oral activity)in a 28-day comparative survival efficacy study in mice systemicallyinfected with Candida albicans. Specifically, male DBA/2 mice (CharlesRiver Laboratories) six weeks of age were dosed with 7.9×10⁴ Candidaalbicans, SLU-1 in 100 μl intravenously via the tail vein in a singledosage on day 0. Treatment began immediately thereafter with 400 μl oralgavage of either 0.5% dextrose, or XMP.445 in 0.5% dextrose at levels ofeither 10 mg/kg or 20 mg/kg every other day for a total of eight times.Amphotericin B (Fungizone®) was administered intravenously at 0.5 mg/kgas a positive control every other day for a total of eight times.Twice-day monitoring (once daily on weekends and holidays) for mortalitywas performed. The animals treated with XMP.445 showed improvements inmortality compared with the dextrose-treated controls, and the animalstreated with XMP.445 at 10 mg/kg showed significant improvement (p-valueof 0.025). The results of this study show that XMP.445 has oralantifungal activity.

EXAMPLE 6 Effect of Miconazole on Fungal and Mammalian Cells Treatedwith DiOC₆(3)

The relative effect of varying concentrations of the antifungal agentmiconazole on fungal C. albicans cells treated with membrane potentialindicator dye DiOC₆(3) [Molecular Probes], mammalian MDCK cells treatedwith DiOC₆(3) and mammalian MDCK cells treated with PI, was assessed asdescribed above in Examples 1 and 2, except that fungal C. albicans ormammalian MDCK cells were resuspended to a concentration of 1×10₆cells/mL.

The results, displayed graphically in FIG. 3 as percent change influorescence mean channel from baseline vs. concentration of miconazole,show that treatment of the C. albicans cells with miconazole andDiOC₆(3) dye resulted in a concentration-dependent increase influorescence intensity with increasing concentration of miconazoledespite a loss of fungal cell viability that occurs at a miconazoleconcentration of less than or equal to about 1 μg/mL. These results showthat treatment with miconazole resulted in accumulation of dye in fungalcells (as measured by increased dye fluorescence intensity) andretention despite reduction of fungal cell viability or fungal celldeath, confirming that other known antifungal agents produce the patternor “fingerprint” characteristic of the BPI-derived peptides XMP.391 andXMP.445. However, miconazole treatment produces a similar pattern ofDiOC6(3) fluorescence increase in mammalian cells, indicating a lack ofselective effect for fungal vs. mammalian cells and a significanttoxicity for mammalian cells. This toxicity is confirmed by the resultsof treating the MDCK cells with miconazole and PI, which produced anincrease in fluorescence intensity indicating cell death.

EXAMPLE 7 Effect of XMP.391 and XMP.445 on Fungal Cells Treated with aVariety of Membrane Potential Indicator Dyes

The relative effect of varying concentrations of BPI-derived peptideXMP.391 on fungal C. albicans cells treated with a variety of membranepotential indicator dyes was evaluated.

The membrane potential indicator dyesJC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide, also called CBIC₂(3)); dihydrorhodamine 123; DiOC₆(3); andMitoTracker® Red CM-H₂Xros (M-7513; C₃₂H₃₃ClN₂) [all dyes obtained fromMolecular Probes] were evaluated at concentrations of 10 μg/mL JC-1, 10μg/mL dihydrorhodamine 123, 10 ng/mL DiOC₆(3) and 1 μg/mL MitoTracker®Red CM-H₂Xros.

Experiments were carried out as described above in Example 1. FL1 (530nm) was used to measure emission of DiOC₆(3); FL2 (585 nm) was used tomeasure emission of rhodamine-123 and MitoTracker® Red CM-H₂Xros; andboth FL1 and FL2 were used to measure the emission ratio ofJC-1/J-aggregate, at an excitation of 488 nm.

Results are displayed graphically in FIG. 4 and show that all four ofthese dyes produce the characteristic pattern of XMP.391 peptideconcentration-dependent dye accumulation and retention in mitochondria.In this experiment, JC-1 and rhodamine 123 produced the highest rise indye fluorescence intensity, primarily because higher concentrations ofthe dye can be used without adverse effects on mitochondrial specificityor fungal cell viability.

Similar results were obtained when experiments were carried out usingXMP.445 and either DiOC₆(3) or MitoTracker® Red CM-H₂Xros dyes.

EXAMPLE 8 Localization of the Accumulation of Membrane PotentialIndicator Dye

The effect of the BPI-derived peptides XMP.391 and XMP.445 on fungalcells treated with a membrane potential indicator dye was furtherexamined using confocal microscopy as follows. Confocal microscopyprovides focused fluorescence detection, one plane or slice at a time,at a focus level that allows unobstructed localization of fluorescencewithin cells. A suspension of 1×10⁶ fungal cells/mL was prepared asdescribed above in Example 1 and 1 mL was dispensed into each of fourEppendorf microcentrifuge tubes. Two μL of 1 mg/mL XMP.445 (in saline)was added to two tubes and all four tubes were incubated for 1 hour at30° C. After the incubation, all tubes were centrifuged in an Eppendorf5415 centrifuge. The supernatant was removed and the pellet resuspendedin 1 mL of SDB. The membrane potential indicator dye DiOC₆(3) (dilutedin SDB from a stock solution of 2 mg/mL in EtOH) was added to one set ofpeptide-treated and untreated tubes to a final concentration of 10ng/mL. The membrane potential indicator dye MitoTracker™ Red CM-H₂Xros(from a freshly prepared solution of 500 μg/mL in DMSO) was added toanother set of peptide-treated and untreated tubes to a finalconcentration of 1 μg/mL. The tubes were incubated at 30° C. for 30 min.After the incubation the tubes were centrifuged and the supernatantremoved. The pellets were resuspended in 100 μL of 30% glycerol/PBSsolution. Three μL from each tube was pipetted onto a microscope slidewith a No. 1 cover glass and placed onto a Zeiss 510 Confocal Microscopethat uses LSM Version 2.01 software. The 10× objective lens was used toisolate and focus on the yeast, then the 100× oil immersion lens wasused to do the confocal microscopy. The following were the approximatesettings for the confocal microscope: Laser: 488 nm for DiOC6(3), or 568nm for MitoTracker™ Red CM-H₂Xros; Scan Mode: Stack; Pixel Dept: 8 bit;Stack Size: 1024×1024×16, 40.7 μm×40.7 μm×3.8 μm; Pixel time: 88 μs;Objective 100× Plan-Apochomat 1.4 oil immersion lens; Beam Splitter: MBSHFT 488 for DiOC₆(3) or 568 for MitoTracker Red™ CM-H₂Xros); DBS1: None;DBS2: Mirror; DBS3: None; Laser power 43%; Filter: BP 505-550 forDiOC₆(3) or LP 585 for MitoTracker™ Red CM-H₂Xros); Pinhole: Ch1 93 μm.The scanned images were stored in TIFF format and is reconstructed andprojected with the LSM 510 software loaded on the Zeiss microscope.

Under these conditions, an average of 10 to 20 cells can be detected perpicture. The effect of XMP.391 was also examined using the sameprocedure. The results showed that in the XMP.391-treated andXMP.445-treated fungal cells, as compared to untreated cells, there wasa visually observable increased fluorescence intensity that waslocalized at the mitochondria.

EXAMPLE 9 Effect of a BPI Protein Product On Bacteria Treated WithDiOC₆(3)

The results of experiments carried out as described above in Example 1using DiOC₆(3) showed that treatment of E. coli J5 with rBPI₂₁ (whichhas in vitro bactericidal activity against this strain) produced anincreasing accumulation of the dye that was retained despite decreasedcell viability, while treatment of E. coli O111 :B4 with rBPI₂₁ (whichdoes not have in vitro bactericidal activity against this strain) didnot produce an accumulation and retention of the dye. Results aredisplayed in FIG. 5, in which the triangles represent treatment of E.coli J5 with rBPI₂₁ and DiOC₆(3), the circles represent treatment of E.coli J5 with rBPI₂₁ and propidium iodide, and the squares representtreatment of E. coli O111 :B4 with rBPI₂₁ and DiOC₆(3)

The results of further experiments with DiOC₆(3) showed that treatmentof E. coli O111 :B4 with XMP.365 [the structure of which is described inU.S. Pat. No. 5,858,974] (which has in vitro bactericidal activityagainst this strain) produced an increasing accumulation of the dye thatwas retained despite decreased cell viability. Results are shown in FIG.6, in which the triangles represent treatment of bacteria with XMP.365and DiOC₆(3), the squares represent treatment of E. coli O111:B4 withXMP.365 and DiBAC(3), and circles represent treatment of E. coli O111:B4with XMP.365 and propidium iodide.

Numerous modifications and variations of the above-described inventionare expected to occur to those of skill in the art. Accordingly, onlysuch limitations as appear in the appended claims should be placedthereon.

2 1 12 PRT Artificial Sequence Modified site at C-Terminus; TheC-Terminus is Amidated 1 Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys1 5 10 2 12 PRT Artificial Sequence position 1 Xaa=D-Lys; position 2Xaa=D-Val; position 11 Xaa=D-Lys; position 12 Xaa=D-Lys 2 Xaa Xaa GlyTrp Leu Ile Gln Leu Phe His Xaa Xaa 1 5 10

What is claimed are:
 1. A method of identifying an antifungal compoundcomprising the steps of: (a) contacting fungal cells with a testcompound and with a membrane potential indicator dye, and (b) detectingan increasing accumulation of said dye and retention of dye despitereduction of fungal cell viability wherein the fungal cells are selectedfrom the group consisting of Candida (including C. albicans, C.tropicalis, C. parapsilosis, C. stellatoidea, C. krusei, C. parakrusei,C. lusitanae, C. pseudotropicalis, C. guilliermondi and C. glabrata),Aspergillus (including A. fumigatus, A. flavus, A. niger, A. nidulans,A. terreus, A. sydowi, A. flavatus, and A. glaucus), Cryptococcus,Histoplasma, Coccidioides, Paracoccidioides, Blastomyces, Basidiobolus,Conidiobolus, Rhizopus, Rhizomucor, Mucor, Absidia, Mortierella,Cunninghamella, Saksenaea, Pseudallescheria, Sporotrichosis, Fusarium,Trichophyton, Trichosporon, Microsporum, Epidermophyton, Scytalidium,Malassezia, Actinomycetes, Sporothrix, Penicillium, Saccharomyces andPneumocystis.
 2. A method of identifying an antibacterial compoundcomprising the steps of: (a) contacting bacterial cells with a testcompound and with a membrane potential indicator dye, and (b) detectingan increasing accumulation of said dye and retention of dye despitereduction of bacterial cell viability wherein the bacterial cells aregram-negative bacteria selected from the group consisting ofAcidaminococcus, Acinetobacter, Aeromonas, Alcaligenes, Bacteroides,Bordetella, Branhamella, Brucella, Calymmatobacterium, Campylobacter,Cardiobacterium, Chromobacterium, Citrobacter, Edwardsiella,Enterobacter, Escherichia, Flavobacterium, Francisella, Fusobacterium,Haemophilus, Klebsiella, Legionella, Moraxella, Morganella, Neisseria,Pasturella, Plesiomonas, Proteus, Providencia, Pseudomonas, Salmonella,Serratia, Shigella, Streptobacillus, Veillonella, Vibrio, and Yersiniaspecies.
 3. A method of identifying an antimicrobial compound comprisingthe steps of: (a) contacting microbial cells with a test compound andwith a membrane potential indicator dye, and (b) detecting an increasingaccumulation of said dye and retention of dye despite reduction ofmicrobial cell viability wherein the microbial cells are gram-positivebacteria selected from the group consisting of Staphylococcus,Streptococcus, Micrococcus, Peptococcus, Peptostreptococcus,Enterococcus, Bacillus, Clostridium, Lactobacillus, Listeria,Erysipelothrix, Propionibacterium, Eubacterium, and Corynebacteriumspecies.
 4. The method of any one of claims 1 through 3 furthercomprising the step of determining microbial cell viability.
 5. Themethod of any one of claims 1 through 3 further comprising the steps of:(a) contacting a mammalian cell with said test compound and with saidmembrane potential indicator dye, and (b) detecting no substantialchange in dye uptake.
 6. The method of any one of claims 1 through 3further comprising the step of assaying said test compound for theability to inhibit growth of microbial cells or to kill microbial cells.7. The method of any one of claims 1 through 3 further comprising thesteps of assaying said test compound for in vivo oral availability ororal activity.
 8. The method of any one of claims 1 through 3 whereinsaid membrane potential indicator dye is DiOC₆(3)(3,3′-dihexyloxacarbocyanine iodide).
 9. The method of any one of claims1 through 3 wherein said membrane potential indicator dye is JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyaninehalide, or CBIC₂(3)).
 10. The method of any one of claims 1 through 3wherein said membrane potential indicator dye is dihydrorhodamine 123.11. The method of any one of claims 1 through 3 wherein said membranepotential indicator dye is MitoTracker® Red CM-H₂Xros (M-7513;C₃₂H₃₃ClN₂O).